Magnitude and kinetics of the human immune cell response associated with severe dengue progression by single-cell proteomics

Abstract

Approximately 5 million dengue virus-infected patients progress to a potentially life-threatening severe dengue (SD) infection annually. To identify the immune features and temporal dynamics underlying SD progression, we performed deep immune profiling by mass cytometry of PBMCs collected longitudinally from SD progressors (SDp) and uncomplicated dengue (D) patients. While D is characterized by early activation of innate immune responses, in SDp there is rapid expansion and activation of IgG-secreting plasma cells and memory and regulatory T cells. Concurrently, SDp, particularly children, demonstrate increased proinflammatory NK cells, inadequate expansion of CD16⁺ monocytes, and high expression of the FcγR CD64 on myeloid cells, yet a signature of diminished antigen presentation. Syndrome-specific determinants include suppressed dendritic cell abundance in shock/hemorrhage versus enriched plasma cell expansion in organ impairment. This study reveals uncoordinated immune responses in SDp and provides insights into SD pathogenesis in humans with potential implications for prediction and treatment.

Fig. 1.. High-dimensional immune profiling delineates disease severity in dengue infection.

(A) Experimental approach. Acute and convalescent PBMC samples were collected from a clinical cohort of DENV-infected patients and healthy controls. Samples were analyzed by mass cytometry using an antibody panel targeting proteins expressed by multiple immune cells. (B) Heatmap of the median-scaled arcsinh-transformed expression (color coded) of lineage molecules (columns) in cell B, NK, myeloid, and T cell populations (rows) spaced by cell type. Cell subtype color annotation is depicted on the right. PBMCs were equally subsampled by clinical status and patient. (C) UMAP generated using all molecules as input colored by cell subtype (B). PBMCs were equally subsampled by clinical status and patient. (D) Analysis approach. Immune features were quantified and compiled into summary statistics for each sample. Distribution tests were performed for each feature for various comparisons. (E) Linear discriminate analysis separating acute patient samples by clinical status. Dots represent individual patients, and ellipses represent 95% confidence intervals (CIs). The manifold was derived using all features significantly different in pairwise comparisons of clinical statuses. (F) Log₂ ratio of median abundances of cell populations (columns) between D and SDp patients of total CD45⁺ cells. Teal bars indicate significance (q < 0.05 and |effect| > 0.5) via Wilcoxon rank sum tests. Q values represent FDR-corrected P values.

Fig. 2.. Immune activation and regulation are simultaneously enriched in acute SDp.

(A and F) Box plots of plasma cell (A) and T_reg (F) fraction of CD45⁺ cells by clinical status. (B) Mean log₂ ratio of patient IgG⁺ plasma cell abundance to IgA⁺ plasma cell abundance by clinical status. Error bars represent SEM. (C to E, I, and K) Box plots of mean Ki-67 (C to E), CTLA-4 (I), and PD-L1 (K) expression in acute (C to E, I, and K) and convalescent samples (D) in the indicated cell populations by clinical status. (G) Proportion of CTLA-4⁺, PD-1⁺ T_regs by clinical status. Quantifications were derived from an equal subsampling of T_regs by clinical status and patient. (H) Scatter plot of mean expression of CD38 versus CTLA-4 in T_regs by patient (dots), colored by clinical status. Fitting line and CI were derived by linear regression. Pearson correlation coefficient (r) and P value are shown. (J) Pearson correlation coefficients (color) in pairwise analysis of mean expression of checkpoint and activation molecules (rows) by T cell population (columns). All correlations are significant (q < 0.05 and |effect| > 0.5) except CTLA-4 × PD-1 and Ki-67 × PD-1 in GD T cells. In box plots, center line signifies median, box signifies interquartile range (IQR), and whiskers signify IQR ± 1.5*IQR. Dots in (A), (C), (D), (F), and (H) represent individual patients. *q < 0.05 and |effect| > 0.5; **q < 0.01 and |effect| > 0.5; ***q < 0.005 and |effect| > 0.5; ^###P < 0.005 and |effect| > 1.0 by Wilcoxon rank sum tests. Q values represent FDR-corrected P values. C, controls; D, dengue; DWS, dengue with warning signs; SDp, SD progressors.

Fig. 3.. Diminished HLA-DR and increased CD64 expression on myeloid cells are hallmarks of SDp.

(A) Difference in median relative abundances of monocyte subtypes (columns) between SDp and D patients of total monocytes. (B) Difference in cohort median of patient mean expression between SDp and D across molecules (rows) in myeloid cell populations (columns). Black boxes depict significance (q < 0.05 and |effect| > 0.05). (C) Box plots of mean HLA-DR expression in the indicated myeloid and NK cell subtypes by clinical status. (D) Box plots of cDC2 fraction of cDCs by clinical status. Dots represent individual patients. (E to H) Scatter plots of mean (E and F) or single-cell CD64 expression versus IgG detection in acute infection on cDC2s (E and G) and monocytes (F and H) by either patient (dots, colored by clinical status) (E and F) or clinical status (G and H). Fitting line and CI in (E) were derived by linear regression. Percent proportion of cells in each quadrant, derived from an equal subsampling of cells by clinical status and patient, are shown in (F) and (G). In box plots, center line signifies median, box signifies IQR, and whiskers signify IQR ± 1.5*IQR. Teal bars (in A) and asterisks (in C and D) indicate significance: *q < 0.05 and |effect| > 0.5; **q < 0.01 and |effect| > 0.5 by Wilcoxon rank sum tests. r and P values (E to G) were calculated by Pearson correlation.

Fig. 4.. Differences in the innate immune response to DENV infection in D and SDp are exaggerated in children.

(A) Effect size in comparison of cell population abundances (dots) in SDp and D in adults (x axis) and children (y axis). Teal and gold indicate significance in adults and children, respectively. (P < 0.05 and |effect| > 1.0). (B and F) Box plots of CD56^dimCD16⁺ NK cell (B) and plasma cell (F) fractions of CD45⁺ cells by clinical status and age. Dots represent individual patients. (C, G, and H) Box plots of mean CD16 (C), Ki-67 (G), and PD-L1 (H) expression in the indicated cells by clinical status and age. Dots represent patients. (D) Proportion of NK cell subtypes in children ordered by CD56^dimCD16⁺ NK cell abundance. Columns represent individual patients, labeled by clinical status. (E) Difference in cohort mean of patient mean HLA-DR expression between SDp and D across cell populations (columns) by age (rows). Black boxes indicate significance (P < 0.05 and |effect| > 1.0). In box plots, center line signifies median, box signifies IQR, and whiskers signify IQR ± 1.5*IQR. ^#P < 0.05 and |effect| > 1.0; ^##P < 0.01 and |effect| > 1.0; ^###P < 0.005 and |effect| > 1.0 by Wilcoxon rank sum tests.

Fig. 5.. Immune cell composition stratifies patients by SD categories.

(A) Log₂ ratio of median abundances of cell populations (columns) between patients with DHF/DSS and OI. Teal bars indicate significance (P < 0.05 and |effect| > 1.0). (B, E, and G) Box plots of cDC1 (B, left), cDC2 (B, right), plasma cell (E), and T_reg (G) fractions of CD45⁺ cells by clinical status. Dots represent individual patients. (C) Proportion of cDC populations ordered by total abundance. Columns represent individual patients, labeled by SD syndrome. (D) Mean CD64 and Ki-67 expression in cDC1s by patient (dots), colored by SD syndrome. (F) Proportion of isotype usage in plasma cells ordered by IgM usage. Columns represent individual patients labeled by SD syndrome. ND, isotype not determined because of low expression. (H) Scatter plot of plasma cell and T_reg abundance by patient (dots), colored by SD syndrome. In box plots, center line signifies median, box signifies IQR, and whiskers signify IQR ± 1.5*IQR. ^#P < 0.05 and |effect| > 1.0; ^##P < 0.01 and |effect| > 1.0; ^###P < 0.005 and |effect| > 1.0 by Wilcoxon rank sum tests. OI, organ impairment; DHF/DSS, dengue hemorrhagic fever/dengue shock syndrome.

Fig. 6.. The temporal switch of innate and adaptive immune activation and concurrent immune regulation is dysregulated in SDp.

(A and E) Difference in cohort mean of patient mean CD38 (A) and CTLA-4 (E) expression between SDp and D across cell populations (rows) by day (columns). Black boxes depict significance (P < 0.05 and |effect| > 1.0). Blue boxes group time points and populations with similar trends. (B) Cohort mean of patient mean CD38 expression in CD8⁺ T EM (top) and CD14⁺ monocytes (bottom) by clinical status and day. (C) Cohort mean of plasma cell abundance (top center), log₂ ratio of IgG⁺ to IgA⁺ plasma cell abundance (top right), and mean CD69 (bottom left) and PD-L1 (bottom right) expression in NK cells by clinical status (color) and day (columns). (D) Cohort mean of patient mean PD-L1 expression (color) by day (columns) and clinical status (rows), separated by cell population (tiles). Each tile is individually scaled. (F) Cohort mean of patient mean CTLA-4 (left) and PD-1 (center) expression in T_regs and abundance of T_regs (right) by clinical status and day. ^#P < 0.05 and |effect| > 1.0; ^##P < 0.01 and |effect| > 1.0; ^###P < 0.005 and |effect| > 1.0 by Wilcoxon rank sum tests. Error bars represent SEM.

Fig. 7.. Proposed hypothetical model for differential immune responses between uncomplicated and SD patients.

Schematic of the kinetics and magnitude of cell subtype abundance and protein marker expression during acute D and SD infections. Most prominent differences in innate and adaptive immune responses are shown. Prominent differences in immune responses between adults and children and SDp with DHF/DSS and OI are also depicted. Black titles depict findings detected in the current study; gray titles depict predicted phenotypes that require further functional validation.